No antivirals or vaccines exist to combat dengue virus (DENV) infection. This is a serious failing since DENV is a recognized Category A bioterrorist agent. In addition, annually DENV causes about1.5 million clinical cases of dengue fever with >500,000 infected individuals developing severe hemorrhagic fever. To address the lack of therapeutics to combat this high-priority and widespread pathogen, we used "virtual screening" to discover putative DEN2V NS3 protease inhibitors. This computationally intensive approach systematically examined a database of small molecules to identify compounds that might interact with either the catalytic or P1 sites of DEN2V NS3 protease. Our computational studies predicted approximately 170 low molecular weight compounds would function as DEN2V NS3 protease inhibitors. Preliminary in vitro assays were done using a small subset of computer-recommended inhibitors and recombinant DEN2V NS3 protease. Significantly, the majority of tested compounds reduced DEN2V NS3 protease activity. Two compounds were further examined in preliminary cell culture experiments; both compounds demonstrated antiviral activity against DEN2V challenge. These compounds had sub-micromolar EC50 values, no apparent cytotoxicity, selectivity indices >150, and no impact on trypsin proteolytic activity. Given our initial success at identifying promising dengue antiviral leads from computer-predicted inhibitors, we hypothesize that additional novel antiviral leads will be found by thoroughly analyzing the entire set of compounds recommended by our computational studies. To test this hypothesis, we will complete in vitro DEN2V NS3 protease assays to determine the Ki and inhibition mechanism of each computer-recommended compound (Specific Aim 1). Compounds with low Ki values will be tested for antiviral activity in cultured cells (Specific Aim 2), and their antiviral mechanism validated using a novel cell-based NS3 protease reporter assay (Specific Aim 3). The specificity of newly discovered inhibitors will be further tested using related serine proteases trypsin and chymotrypsin, and blood-clotting assays. Since hemorrhagic complications often accompany DENV infection, the blood-clotting assay is prudent to ensure dengue protease inhibitors do not interfere with the blood coagulation cascade (Specific Aim 4). Successful completion of this project will identify and characterize new antiviral leads for further development as therapeutics for dengue virus infection. In addition, determining the mechanistic basis and specificity of these lead antivirals will provide a solid foundation for future structure-based antiviral optimization studies to produce therapeutics effective against related flaviviruses, including West Nile, Yellow Fever, and Hepatitis C viruses.